Stabilization ponds are commonly used for wastewater treatment in developing countries. However, little is known about the microorganisms present in such systems. A combination of denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) was used for qualitative and quantitative analysis of spatial and seasonal variations of microbial diversity and community structure. Samples were collected both in winter and summer from three depth of the bulk water and from the bottom sludge of a shallow stabilization pond treating municipal wastewater of the city of Cajati, Brazil; in addition, 27 stations across the pond were sampled at an intermediate depth. DGGE revealed little variation of the bacterial diversity among water depths and stations, while the bacterial communities differed between water and sludge, and between seasons. CARD-FISH successfully detected 55-85% and 40-50% of all cells in the water and sludge samples, respectively, and generally confirmed the DGGE results. The bacterial community was dominated by unicellular cyanobacteria, followed by either Beta- or Alphaproteobacteria, depending on the season. Other bacterial groups detected were Gammaproteobacteria, Planctomycetes, and the Cytophaga-Flavobacterium and Chloroflexus groups. Archaea accounted for < 3% in the water and >5% in the sludge, probably due to the presence of methanogens in the sludge. The absence of depth- and site-related variation in microbial community structure indicates that the stabilization pond is well mixed, which is also reflected in the physical-chemical data. In contrast, strong seasonal variation was observed, which influenced the functioning of the stabilization pond.